Isolated nucleic acids encoding methylenetetrahydrofolate dehydrogenase enzymes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a tetrahydrofoláte metabolism enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the tetrahydrofolate metabolism enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the tetrahydrofolate metabolism enzyme in a transformed host cell.

[0001] This application claims the benefit of U.S. Provisional Application No. 60/092,869, filed Jul. 15, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding tetrahydrofolate metabolism enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Tetrahydrofolic acid and its derivatives N⁵,N¹⁰-methylenetetrahydrofolate, N⁵,N¹⁰-methenyltetrahydrofolate, N¹⁰-formyltetrahydrofolate, and N⁵-methyltetra-hydrofolate are the biologically active forms of folic acid, oxidized form of tetrahydrofolate (THF). The tetrahydrofolates are coenzymes which are not enzyme-bound and are specialized cosubstrates for a variety of enzymes involved in one-carbon metabolism. Tetrahydrofolate (THF) is a 6-methylpterin derivative linked to p-aminobenzoic acid and glutamic acid residues. Its function is to transfer C1 units in several oxidation states. The C1 units are convalently attached to THF at its N5 and/or N10 positions and enter into the THF pool through the conversion of serine to glycine by serine hydroxymethyl transferase and the cleavage of glycine by glycine synthase. A C1 unit in the THF pool can have several outcomes: it may be used in the conversion of the deoxynucleotide dUMP to dTMP by thymidylate synthase, it may be reduced for the synthesis of methionine, or it may oxidized for the use in the synthesis of purines, since the purines ring of ATP is involved in histidine biosynthesis.

[0004] There are several enzymes involved in tetrahydrofolate metabolism five of which are, methylenetetrahydrofolate dehydrogenase (NADP+), 5,10-methylenetetrahydrofolate reductase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, glutamate formyltransferase, or formyltetrahydrofolate deformylase. Methylenetetrahydrofolate dehydrogenase (NADP+) is an oxidoreductase which acts on the CH—NH group of donors with NAD+ or NADP+ as acceptor. In eucaryotes it occurs as a trifunctional enzyme also having methenyltetra-hydrofolate cyclohydrolase (EC 3.5.4.9) and formyltetrahydrofolate synthase (EC 6.3.4.3) activity. In some prokaryotes it occurs as a bifunctional enzyme also having methenyltetrahydrofolate cyclohydrolase activity (EC 3.5.4.9). This trifunctional enzyme consists of two major domains: an aminoterminal part, containing the methylene-THF dehydrogenase and methenyl-THF cyclohydrolase activities and a larger formyl-THF synthetase domain.

[0005] 5,10-Methylenetetrahydrofolate reductase (EC 1.7.99.5) (MTHFR) plays a role in the synthesis of methionine (West et al, (1993) J. Biol. Chem. 268:153-160 and D'Ari et al. (1991) J. Biol. Chem. 266:23953-23958). S-adenosylmethionine (SAM) an important methyl group donor for many biosynthetic methylation reactions in plants. SAM is formed from methionine by SAM synthetase. Transfer of the methyl group from SAM to an acceptor molecule results in the formation of S-adenosylhomocysteine, which is then hydrolyzed to homocysteine. Methionine is regenerated from homocysteine by methyl group transfer from 5-methyltetrahydrofolate. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. The heavy demand in plant cells for methyl groups derived from SAM necessitate a rapid recycling of S-adenosylhomocysteine, and thus a heavy demand for 5-methyltetrahydrofolate produced by MTHFR.

[0006] 3-Methyl-2-oxobutanoate hydroxymethyltransferase (EC 2.1.2.11) is the first enzyme in the pantothenate biosynthetic pathway. This enzyme catalyses the conversion of 5,10-methylenetetrahydrofolate and 3-methyl-2-oxobutanoate to tetrahydrofolate and 2-dehydropantoate. Pantothenate is a vitamin required in the diet of animals. It is used in the synthesis of coenzyme A, which in turn, is used in many important enzyme reactions in many pathways, e.g., fatty acid biosynthesis. The production of high levels fatty acids, which require coenzyme A for their synthesis, might be stimulated by production of higher levels of coenzyme A, which in turn would require increased production of pantothenate. Another use might be for the increased production of pantothenate in plants in order to purify this vitamin for sale.

[0007] Glutamate formyltransferase (EC 2.1.2.5) catalyses the transfer of a formyl group from 5-formyltetrahydrofolate to L-glutamate. This enzyme serves to channel one-carbon units from formiminoglutamate to the folate pool.

[0008] Lastly, formyltetrahydrofolate deformylase (EC 3.5.1.10) catalyses the formation of formate and tetrahydrofolate from 10-formyltetrahydrofolate and water. 10-Formyltetra-hydrofolate is required in de novo purine biosynthesis and histidine biosynthesis.

[0009] Because these enzymes are involved in tetrahydrofolate metabolism, amino acid synthesis, fatty acid biosynthesis and de novo synthesis of purines inhibition of their activity may be lethal, thus suggesting that they would be attractive herbicide targets. Thus production of these plant enzymes in bacteria for use in a high throughput screen for chemical inhibitors would be desirable. Alternatively, overproduction of these enzymes in transgenic plants could be used to enhance the production of many secondary metabolites, amino acids, purine nucleic acids and vitamins. Accordingly, the availability of nucleic acid sequences encoding all or a portion of an enzyme involved in tetrahydrofolate metabolism would facilitate studies to better understand tetrahydrofolate metabolism in plants, provide genetic tools to enhance the production of secondary metabolites, amino acids and vitamins. These enzymes may also provide targets to facilitate design and/or identification of inhibitors tetrahydrofolate metabolism that may be useful as herbicides.

SUMMARY OF THE INVENTION

[0010] The instant invention relates to isolated nucleic acid fragments encoding tetrahydrofolate metabolism enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetra-hydrofolate dehydrogenase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyl-transferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase.

[0011] An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a tetrahydrofolate metabolism enzyme selected from the group consisting of 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase and methylenetetrahydrofolate dehydrogenase.

[0012] In another embodiment, the instant invention relates to a chimeric gene encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetra-hydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.

[0013] In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formimino-transferase or methylenetetrahydrofolate dehydrogenase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.

[0014] An additional embodiment of the instant invention concerns a method of altering the level of expression of a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylene-tetrahydrofolate dehydrogenase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase in the transformed host cell.

[0015] An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase.

[0016] A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a 3-methyl-2-oxobutanoate hydroxymethyl-transferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase; glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenete-trahydrofolate dehydrogenase in the transformed host cell; (c) optionally purifying the 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase expressed by the transformed host cell; (d) treating the 3-methyl-2-oxobutanoate hydroxymethyl-transferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase with a compound to be tested; and (e) comparing the activity of the 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetra-hydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase that has been treated with a test compound to the activity of an untreated 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, thereby selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

[0017] The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.

[0018] Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. TABLE 1 Tetrahydrofolate Metabolism Enzymes SEQ ID NO: (Nucleo- (Amino Protein Clone Designation tide) Acid) 3-Methyl-2-oxobutanoate cho1c.pk001.i4 1 2 hydroxymethyltransferase Formyltetrahydrofolate Contig composed of: 3 4 deformylase cpd1c.pk010.f6 p0127.cntbp47r p0127.cntbp48r Formyltetrahydrofolate rls72.pk0011.d1 5 6 deformylase Formyltetrahydrofolate wr1.pk0075.c11 7 8 deformylase Glutamate formimino- rls6.pk0086.g2 9 10 transferase Glutamate formimino- sfl1n.pk003.o10 11 12 transferase Glutamate formimino- wre1n.pk0041.e8 13 14 transferase Methylenetetrahydrofolate Contig composed of: 15 16 dehydrogenase cbn10.pk0021.f6 cbn2.pk0047.b3 cbn2n.pk0012.g11 cc71.pk0002.f2 cco1n.pk0041.d9 cen3n.pk0027.c12 cen3n.pk0128.d9 cr1n.pk0196.f11 cta1n.pk0073.g9 Methylenetetrahydrofolate rlr6.pk0059.b1 17 18 dehydrogenase Methylenetetrahydrofolate ses2w.pk0009.d8 19 20 dehydrogenase Methylenetetrahydrofolate Contig composed of: 21 22 dehydrogenase wdk1c.pk012.f19 wl1.pk0006.h11 wle1n.pk0031.g8 wr1.pk0101.c9 wr1.pk0118.c6 wre1n.pk170.f4 wre1n.pk170.g10

[0019] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0020] In the context of this disclosure, a number of terms shall be utilized. As used herein, a “nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0021] As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

[0022] As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

[0023] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0024] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

[0025] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Preferred are those nucleic acid fragments whose nucleotide sequences encode amino acid sequences that are 80% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are 95% identical to the amino acid sequences reported herein. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0026] A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0027] “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0028] “Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0029] “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0030] “Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0031] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0032] The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Molecular Biotechnology 3:225).

[0033] The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0034] “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptide by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0035] The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0036] The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

[0037] “Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0038] “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0039] A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

[0040] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

[0041] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

[0042] Nucleic acid fragments encoding at least a portion of several tetrahydrofolate metabolism enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0043] For example, genes encoding other 3-methyl-2-oxobutanoate hydroxymethyl-transferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase enzymes, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0044] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989) Science 243:217). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

[0045] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1; Maniatis).

[0046] The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of tetrahydrofolate metabolism in those cells.

[0047] Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

[0048] Plasmid vectors comprising the instant chimeric gene can then constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0049] For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.

[0050] It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

[0051] Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U. S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

[0052] The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppresion technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds, and is not an inherent part of the invention. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

[0053] The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded tetrahydrofolate metabolism enzyme. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 9).

[0054] Additionally, the instant polypeptides can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in tetrahydrofolate metabolism. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design.

[0055] All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0056] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0057] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0058] In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Research 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0059] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0060] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell 7:75). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptides. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptides can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

EXAMPLES

[0061] The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0062] cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library Tissue Clone cbn2 Corn (Zea mays L.) developing kernel two days after cbn2.pk0047.b3 pollination cbn2n Corn (Zea mays L.) developing kernel two days after cbn2n.pk0012.g11 pollination* cbn10 Corn (Zea mays L.) developing kernel (embryo and cbn10.pk0021.f6 endosperm; 10 days after pollination) cc71 Corn (Zea mays L.) type II callus tissue, undifferentiated cc71.pk0002.f2 cco1n Corn (Zea mays L.) cob of 67 day old plants grown in cco1n.pk0041.d9 green house* cen3n Corn (Zea mays L.) endosperm stage 3 (20 days after cen3n.pk0027.c12 pollination)* cen3n.pk0128.d9 cho1c Corn (Zea mays L.) embryo 20 days after pollination cho1c.pk001.i4 cpd1c Corn (Zea mays L.) pooled BMS treated with chemicals cpd1c.pk010.f6 related to protein kinases** cr1n Corn (Zea mays L.) root from 7 day seedlings grown in light* cr1n.pk0196.f11 cta1n Corn (Zea mays L.) tassel* cta1n.pk0073.g9 p0127 Corn (Zea mays L.) nucellus tissue, 5 days after silking* p0127.cntbp47r p0127.cntbp48r rlr6 Rice (Oryza sativa L.) leaf (15 days after germination) 6 hrs rlr6.pk0059.b1 after infection of Magnaporthe grisea strain 4360-R-62 (AVR2-YAMO); Resistant rls6 Rice (Oryza sativa L.) leaf (15 days after germination) 6 hrs rls6.pk0086.g2 after infection of Magnaporthe grisea strain 4360-R-67 (avr2-yamo); Susceptible rls72 Rice (Oryza sativa L.) leaf (15 days after germination) rls72.pk0011.d1 72 hours after infection of Magnaporthe grisea strain 4360-R-67 (avr2-yamo); Susceptible ses2w Soybean (Glycine max L.) embryogenic suspension 2 weeks ses2w.pk0009.d8 after subculture sfl1n Soybean (Glycine max L,) immature flower sfl1n.pk003.o10 wdk1c Wheat (Triticum aestivum L.) developing kernel, 3 days wdk1c.pk012.f19 after anthesis wl1 Wheat (Triticum aestivum L.) leaf 7 day old seedling, light wl1.pk0006.h11 grown wle1n Wheat (Triticum aestivum L.) leaf 7 day old etiolated wle1n.pk0031.g8 seedling wr1 Wheat (Triticum aestivum L.) root; 7 day old etiolated wr1.pk0075.c11 seedling wr1.pk0101.c9 wr1.pk0118.c6 wre1n Wheat (Triticum aestivum L.) root; 7 day old etiolated wre1n.pk0041.e8 seedling wre1n.pk170.g10 wre1n.pk170.f4

[0063] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP* XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP* XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0064] cDNA clones encoding tetrahydrofolate metabolism enzymes were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding 3-Methyl-2-oxobutanoate hydroxymethyltransferase

[0065] The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to 3-methyl-2-oxobutanoate hydroxymethyltransferase from Emericella nidulans (NCBI Identifier No. gi 5001995). Shown in Table 3 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), or contigs assembled from two or more ESTs (“Contig”): TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to Emericella nidulans 3-Methyl-2-oxobutanoate hydroxymethyltransferase Clone Status BLAST pLog Score to (gi 5001995) cho1c.pk001.i4 EST 70.00

[0066] The data in Table 4 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2 and the Emericella nidulans sequence. TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Emericella nidulans 3-Methyl-2-oxobutanoate hydroxymethyltransferase Percent Identity to SEQ ID NO. (gi 5001995) 2 39%

[0067] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a 3-methyl-2-oxobutanoate hydroxymethyltransferase. These sequences represent the first corn sequences encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase.

Example 4 Characterization of cDNA Clones Encoding Formyltetrahydrofolate Deformylase

[0068] The BLASTX search using the EST sequences from clones listed in Table 5 revealed similarity of the polypeptides encoded by the cDNAs to formyltetrahydrofolate deformylase from Aquifex aeeolicus (NCBI Identifier No. gi 2984098) and Corynebacterium sp. (NCBI Identifier No. gi 2500006). Shown in Table 5 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), or contigs assembled from two or more ESTs (“Contig”): TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous to Aquifex aeeolicus and Corynebacterium sp. Formyltetrahydrofolate Deformylase Clone Status BLAST pLog Score Contig composed of: Contig 33.30 (gi 2984098) cpd1c.pk010.f6 p0127.cntbp47r p0127.cntbp48r rls72.pk0011.d1 EST 75.00 (gi 2500006) wr1.pk0075.c11 EST  5.70 (gi 2984098)

[0069] The data in Table 6 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:4, 6 and 8 and the Aquifex aeeolicus and Corynebacterium sp. sequences. TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Aquifex aeeolicus and Corynebacterium sp. Formyltetrahydrofolate Deformylase SEQ ID NO. Percent Identity to 4 46% (gi 2984098) 6 49% (gi 250006) 8 30% (gi 2984098)

[0070] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a formyltetrahydrofolate deformylase. These sequences represent the first corn, rice and wheat sequences encoding formyltetrahydrofolate deformylase.

Example 5 Characterization of cDNA Clones Encoding Glutamate Formiminotransferase

[0071] The BLASTX search using the EST sequences from clones listed in Table 7 revealed similarity of the polypeptides encoded by the cDNAs to glutamate formiminotransferase from Aquifex aeeolicus (NCBI Identifier No. gi 1706872). Shown in Table 7 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), or contigs assembled from two or more ESTs (“Contig”): TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous to Aquifex aeeolicus Glutamate Formiminotransferase Clone Status BLAST pLog Score to (gi 1706872) rls6.pk0086.g2 EST 12.05 sfl1.pk003.o10 EST 7.00 wre1n.pk0041.e8 EST 13.30

[0072] The data in Table * represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs: 10, 12 and 14 and the Aquifex aeeolicus sequence. TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Aquifex aeeolicus Glutamate Formiminotransferase SEQ ID NO. Percent Identity to (gi 1706872) 10 20% 12 24% 14 20%

[0073] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a glutamate formiminotransferase. These sequences represent the first rice, soybean and wheat sequences encoding glutamate formiminotransferase.

Example 6 Characterization of cDNA Clones Encoding Methylenetetrahydrofolate Dehydrogenase

[0074] The BLASTX search using the EST sequences from clones listed in Table 9 revealed similarity of the polypeptides encoded by the cDNAs to methylenetetrahydrofolate dehydrogenase from Pisum sativum (NCBI Identifier No. gi 4103987). Shown in Table 9 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), or contigs assembled from two or more ESTs (“Contig”): TABLE 9 BLAST Results for Sequences Encoding Polypeptides omologous to Pisum sativum Methylenetetrahydrofolate Dehydrogenase Clone Status BLAST pLog Score to (gi 1706872) Contig composed of: Contig 130.00 cbn10.pk0021.f6 cbn2.pk0047.b3 cbn2n.pk0012.g11 cc71.pk0002.f2 cco1n.pk0041.d9 cen3n.pk0027.c12 cen3n.pk0128.d9 cr1n.pk0196.f11 cta1n.pk0073 g9 rlr6.pk0059.b1 EST 113.00 ses2w.pk0009.d8 EST 88.70 Contig composed of: Contig 82.15 wdk1c.pk012.f19 wl1.pk0006.h11 wle1n.pk0031.g8 wr1.pk0101.c9 wr1.pk0118.c6 wre1n.pk170.f4 wre1n.pk170.g10

[0075] The data in Table 10 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:16, 18 and 20 and the Pisum sativum sequence. TABLE 10 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Pisum sativum Methylenetetrahydrofolate Dehydrogenase SEQ ID NO. Percent Identity to (gi 1706872) 16 78% 18 66% 20 63% 22 55%

[0076] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a methylenetetrahydrofolate dehydrogenase. These sequences represent the first corn, rice and soybean sequences encoding methylenetetrahydrofolate dehydrogenase.

Example 7 Expression of Chimeric Genes in Monocot Cells

[0077] A chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3′ region.

[0078] The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0079] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0080] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0081] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0082] Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0083] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 8 Expression of Chimeric Genes in Dicot Cells

[0084] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

[0085] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

[0086] Soybean embroys may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0087] Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0088] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0089] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptides and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0090] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0091] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0092] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 9 Expression of Chimeric Genes in Microbial Cells

[0093] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0094] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0095] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 10 Evaluating Compounds for Their Ability to Inhibit the Activity of Tetrahydrofolate Metabolism Enzymes

[0096] The polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 9, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines. The instant polypeptides may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase (“GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide (“(His)₆”). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.

[0097] Purification of the instant polypeptides, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the instant polypeptides are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)₆ peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin.

[0098] Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the instant polypeptides disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity.

1 22 1 560 DNA Zea mays unsure (442) n is a, c, g or t 1 gctccccaca ggcccacacc aaccaactct ctctgcctcg gtccctccgc ccgtacgcgc 60 gacgagagac acgatgcggc ggtccctccc cctcctcctc gcgcggcagg tggcgcggca 120 gcggcggctg agcaacgtgc cggagtccac cgtctacggg ggcccacgcc cgcaggagtc 180 ctcggcggcg cggcgcgtga cggtgaccac actccgtggg aagcaccgcc gcggggagcc 240 catcaccgtc gtcaccgcct acgactaccc ctcggcggtc cacgtcgact ccgccggcat 300 cgacgtctgc ctcgtcgggg actccgccgc catggtcgtc cacggccacg acaccacgct 360 ccccatcacg ctcgacatca tgctcgaagc actgccgcgc cgttggcccg gggcgcgccg 420 cgcccgctcc tcgtcgggga tntccaattc ggctgctaca atccttcggc gccaagctgt 480 tgattaaccg ttaaggtctc aaggaagtgg atggtgcatn aactggaagg ggtgccatca 540 ggttattgaa caaaggtatt 560 2 375 PRT Zea mays 2 Pro Thr Gly Pro His Gln Pro Thr Leu Ser Ala Ser Val Pro Pro Pro 1 5 10 15 Val Arg Ala Thr Arg Asp Thr Met Arg Arg Ser Leu Pro Leu Leu Leu 20 25 30 Ala Arg Gln Val Ala Arg Gln Arg Arg Leu Ser Asn Val Pro Glu Ser 35 40 45 Thr Val Tyr Gly Gly Pro Arg Pro Gln Glu Ser Ser Ala Ala Arg Arg 50 55 60 Val Thr Val Thr Thr Leu Arg Gly Lys His Arg Arg Gly Glu Pro Ile 65 70 75 80 Thr Val Val Thr Ala Tyr Asp Tyr Pro Ser Ala Val His Val Asp Ser 85 90 95 Ala Gly Ile Asp Val Cys Leu Val Gly Asp Ser Ala Ala Met Val Val 100 105 110 His Gly His Asp Thr Thr Leu Pro Ile Thr Leu Asp Ile Met Leu Glu 115 120 125 His Cys Arg Ala Val Ala Arg Gly Ala Pro Arg Pro Leu Leu Val Gly 130 135 140 Asp Leu Pro Phe Gly Cys Tyr Glu Ser Ser Ala Ala Gln Ala Val Asp 145 150 155 160 Ser Ala Val Arg Val Leu Lys Glu Gly Gly Met Asp Ala Ile Lys Leu 165 170 175 Glu Gly Gly Ala Pro Ser Arg Ile Thr Ala Ala Lys Ala Ile Val Glu 180 185 190 Ala Gly Ile Ala Val Met Gly His Val Gly Leu Thr Pro Gln Ala Ile 195 200 205 Ser Val Leu Gly Gly Phe Arg Pro Gln Gly Lys Thr Val Asp Ser Ala 210 215 220 Ile Lys Val Val Glu Thr Ala Leu Ala Leu Gln Glu Ala Gly Cys Phe 225 230 235 240 Ser Val Val Leu Glu Cys Val Pro Ala Pro Val Ala Ala Ala Ala Thr 245 250 255 Ser Ala Leu Lys Ile Pro Thr Ile Gly Ile Gly Ala Gly Pro Phe Cys 260 265 270 Ser Gly Gln Val Leu Val Tyr His Asp Leu Leu Gly Met Leu Gln His 275 280 285 Pro His His Ala Lys Val Thr Pro Lys Phe Cys Lys Gln Phe Gly Asn 290 295 300 Val Gly Asp Val Ile Asn Lys Ala Leu Ser Glu Tyr Lys Gln Glu Val 305 310 315 320 Glu Ala Gln Ala Phe Pro Gly Pro Ser His Thr Pro Tyr Lys Ile Thr 325 330 335 Pro Thr Asp Val Asp Gly Phe Ala Asp Ala Leu Gln Lys Met Gly Leu 340 345 350 Ser Asp Ala Ala Asp Ala Ala Ala Ala Ala Ala Glu Asn Arg Glu Lys 355 360 365 Gly Gly Glu Pro Asn Gly Glu 370 375 3 652 DNA Zea mays unsure (595) n is a, c, g or t 3 gctctataga tggcaagaag gcaggcttcc agtacacatt aattgtgtca ttagcaacca 60 tgatagacca cagataatca tgtacggcgt tttctccaga ggcatggaat cccataccac 120 tacttaccca ccgcacctgc caacaaaaga gagaaagaga tcttggaatt gatccagggt 180 acagattttg ttgtgctggc aagatacatg cagatactct cagaaaacct gttaaaagca 240 tatggtaaag acattatcaa tattcatcat ggccttcttc cctcatttaa gggagggaat 300 ccttcaagac aggccttcag tgctggggtg aagttaatcg gggcaactag ccatttcgtt 360 actccagaac ttgatgctgg gccaatcatt gaacagatgg ttgaacgagt ctctcaccga 420 gacacgttac agagttttgt tgtcaagtct gagaaccttg agaagcagtg cttaacagaa 480 gctatcaagt catattgcga gcttcgtgtc taccatatga actcaggaag actgtcgtgg 540 tctgatctga gcttccttta ttttctggct taattggact tttatatggg attgntaaaa 600 tgaaattttt aactttaaat atattcattc ccctcgacaa ttattttaag gg 652 4 169 PRT Zea mays 4 Leu Tyr Arg Trp Gln Glu Gly Arg Leu Pro Val His Ile Asn Cys Val 1 5 10 15 Ile Ser Asn His Asp Arg Val Arg Arg Phe Leu Gln Arg His Gly Ile 20 25 30 Pro Tyr His Tyr Leu Pro Thr Ala Pro Ala Asn Lys Arg Glu Lys Glu 35 40 45 Ile Leu Glu Leu Ile Gln Gly Thr Asp Phe Val Val Leu Ala Arg Tyr 50 55 60 Met Gln Ile Leu Ser Glu Asn Leu Leu Lys Ala Tyr Gly Lys Asp Ile 65 70 75 80 Ile Asn Ile His His Gly Leu Leu Pro Ser Phe Lys Gly Gly Asn Pro 85 90 95 Ser Arg Gln Ala Phe Ser Ala Gly Val Lys Leu Ile Gly Ala Thr Ser 100 105 110 His Phe Val Thr Pro Glu Leu Asp Ala Gly Pro Ile Ile Glu Gln Met 115 120 125 Val Glu Arg Val Ser His Arg Asp Thr Leu Gln Ser Phe Val Val Lys 130 135 140 Ser Glu Asn Leu Glu Lys Gln Cys Leu Thr Glu Ala Ile Lys Ser Tyr 145 150 155 160 Cys Glu Leu Arg Val Tyr His Met Asn 165 5 563 DNA Oryza sativa unsure (505) n is a, c, g or t 5 tacagcgcca tcagttcaac ctgacaatcg gcgaagaaac tcaattgccg tccccaccaa 60 ttctgaaagt ccaccacttt cggcccaccc ccctccaacc ccaaattcaa caatcatggc 120 cgccaacgac gaccacatcc tgacgctgtc atgcccggac aagccgggca tcgtccacgc 180 cgtgactggc atctttgcct cgcggtcggt caacattctt gacctgaagc agttctccga 240 cacggggtcg caaaagttct tcatgcgggt gcactttggc ccagtggccg agacggcgga 300 cctctctgcc gacttctcgg ctctggcgtc gcagtacgac cccatgacct gggacatccg 360 gcccgtggcg caaaagacgc gcgtcctgat atggtgtcaa gatcggcact gtctcaacga 420 cctgctgttc cgcgcccaga gcggccgcct cgccgtcact ggcctcatcg tgtcaacacc 480 cgacttgcgc cctggcgcag cacgngtcan tcgcactgcc gtcacaagaa caagaccaca 540 ggagnagaat ccaactgcaa gac 563 6 278 PRT Oryza sativa 6 His Ile Leu Thr Leu Ser Cys Pro Asp Lys Pro Gly Ile Val His Ala 1 5 10 15 Val Thr Gly Ile Phe Ala Ser Arg Ser Val Asn Ile Leu Asp Leu Lys 20 25 30 Gln Phe Ser Asp Thr Gly Ser Gln Lys Phe Phe Met Arg Val His Phe 35 40 45 Gly Pro Val Ala Glu Thr Ala Asp Leu Ser Ala Asp Phe Ser Ala Leu 50 55 60 Ala Ser Gln Tyr Asp Pro Met Thr Trp Asp Ile Arg Pro Val Ala Gln 65 70 75 80 Lys Thr Arg Val Leu Ile Met Val Ser Lys Ile Gly His Cys Leu Asn 85 90 95 Asp Leu Leu Phe Arg Ala Gln Ser Gly Arg Leu Ala Val Asp Val Ala 100 105 110 Leu Ile Val Ser Asn His Pro Asp Phe Ala Pro Leu Ala Ala Ser His 115 120 125 Gly Val Glu Phe Arg His Leu Pro Val Thr Lys Glu Thr Lys Thr Gln 130 135 140 Gln Glu Glu Glu Ile Leu Lys Leu Ala Lys Glu Arg Asp Val Glu Leu 145 150 155 160 Ile Val Leu Ala Arg Tyr Met Gln Val Leu Ser Pro Thr Leu Cys Glu 165 170 175 Ala Met Ser Gly Arg Ile Ile Asn Ile His His Ser Phe Leu Pro Ser 180 185 190 Phe Lys Gly Ala Lys Pro Tyr His Gln Ala Tyr Asp Arg Gly Val Lys 195 200 205 Ile Ile Gly Ala Thr Ala His Phe Val Thr Ala Asp Leu Asp Glu Gly 210 215 220 Pro Ile Ile Glu Gln Arg Ile Ser Arg Val Asp His Gly Met Thr Pro 225 230 235 240 Lys Gln Leu Val Asp Glu Gly Ser Ser Ile Glu Ala Leu Val Leu Gly 245 250 255 Ala Ala Val Gln Trp Phe Ala Glu Arg Arg Val Phe Leu Asn Asn Ser 260 265 270 Lys Thr Val Val Phe Asn 275 7 594 DNA Triticum aestivum unsure (358) n is a, c, g or t 7 caacccgagg ctatggccgc gcgacgtgct tcgcgccgac ttcctccgcc tgtcggactg 60 cttcagcgcg caaaaatcca ctgtgcgagt acctgacatc gaccccaagt acaagattgc 120 agtcctcgct tcgaagcagg accattgtct gtttgacttg ctgcatagat ggcaagaagg 180 caggcttcca gttgacattc attgtgtgat aagcaaccat gatcgacctg tagataacca 240 tgtgatgcgt tttcttcaag aggcacgaaa tcccctatca ttacttacca acgacttcct 300 gggaataaaa gggaacaaga gatattagaa ttgattgaag atacagattt tgttgtgntg 360 ggcaagatat gcangtaatg tcngaaactt ccttaaacat atgggaaaga tattattata 420 tcacaaggct ccttcctcng tcnaaaggag gatcctctag naggctcaat gctgggtnaa 480 ttgattggtg cacnaccatt tgtacccana cttagcgggc aacatngacc aaggtnaacg 540 gtcccanagg aaattaanac ttgtntnatc tgaaactngg aacatccaca aann 594 8 70 PRT Triticum aestivum 8 Pro Arg Asp Val Leu Arg Ala Asp Phe Leu Arg Leu Ser Asp Cys Phe 1 5 10 15 Ser Ala Gln Lys Ser Thr Val Arg Val Pro Asp Ile Asp Pro Lys Tyr 20 25 30 Lys Ile Ala Val Leu Ala Ser Lys Gln Asp His Cys Leu Phe Asp Leu 35 40 45 Leu His Arg Trp Gln Glu Gly Arg Leu Pro Val Asp Ile His Cys Val 50 55 60 Ile Ser Asn His Asp Arg 65 70 9 543 DNA Oryza sativa unsure (381) n is a, c, g or t 9 gttctaacgc tgcaagctct acatttctga aagccagaat gccaaggttg ttgatgcaat 60 cactcgcatt ggccagaaag accctgaggt tgttttgctc agcaagttcg aagatgacca 120 ctacaaccgt gtccgttaca cgcttgcgtc ttatatcatc aacgagaact caactggtga 180 agtgaaattt agcccaatga ggcgagtatt gttggagatg attgagaaag cgttttcaac 240 cataaacctt gaaacgcaca ctgggaactc acccaaggat tggagtcatt gatgacatgt 300 ccttccaccc cttgaatcaa gccacaatgg aagatgctgc tcaactggct aagactgtgg 360 cctctgacat tggcaacttc ntacaagtcc cagtatcctg tatggagcag cacaccccac 420 tggcaaacct gtgactgcan tacggcgtna actgggctac ttccanccan attcatgggc 480 atccaatggg atgggtcagg taccccntga tatctgcggg naanccagat aagggccagt 540 ttg 543 10 296 PRT Oryza sativa 10 Arg Cys Lys Leu Tyr Ile Ser Glu Ser Gln Asn Ala Lys Val Val Asp 1 5 10 15 Ala Ile Thr Arg Ile Gly Gln Lys Asp Pro Glu Val Val Leu Leu Ser 20 25 30 Lys Phe Glu Asp Asp His Tyr Asn Arg Val Arg Tyr Thr Leu Ala Ser 35 40 45 Tyr Ile Ile Asn Glu Asn Ser Thr Gly Glu Val Lys Phe Ser Pro Met 50 55 60 Arg Arg Val Leu Leu Glu Met Ile Glu Lys Ala Phe Ser Thr Ile Asn 65 70 75 80 Leu Glu Thr His Thr Gly Thr His Pro Arg Ile Gly Val Ile Asp Asp 85 90 95 Met Ser Phe His Pro Leu Asn Gln Ala Thr Met Glu Asp Ala Ala Gln 100 105 110 Leu Ala Lys Thr Val Ala Ser Asp Ile Gly Asn Phe Leu Gln Val Pro 115 120 125 Val Phe Leu Tyr Gly Ala Ala His Pro Thr Gly Lys Pro Val Thr Ala 130 135 140 Val Arg Arg Glu Leu Gly Tyr Phe Gln Pro Asn Tyr Met Gly Ile Gln 145 150 155 160 Trp Met Gly Gln Val Leu Pro Asp Ile Leu Pro Val Lys Pro Asp Glu 165 170 175 Gly Pro Asp His Val Ser Arg Glu Arg Gly Ala Ile Met Ile Gly Ala 180 185 190 Ala Pro Leu Pro Leu Asn Tyr Asn Val Pro Val Leu Ser Lys Asp Ile 195 200 205 Pro Thr Ile Arg Arg Ile Thr Arg Arg Val Thr Gly Arg Gly Gly Gly 210 215 220 Leu Pro Thr Val Gln Ala Leu Ala Leu Ser His Gly Asp Asp Cys Thr 225 230 235 240 Glu Ile Ala Cys Phe Leu Asp Pro Asp His Val Ser Ala Asp Gln Val 245 250 255 Gln Gln Gln Val Glu Gln Ile Ala Ala Glu Gln Gly Leu Glu Val Glu 260 265 270 Lys Gly Tyr Phe Thr Asp Phe Ser Lys Asp Ala Met Leu Glu Lys Tyr 275 280 285 Phe Lys Ile Val Leu Ser Val Asp 290 295 11 468 DNA Glycine max unsure (398) n is a, c, g or t 11 ggaaatccca tatacagccc cttgcaccaa actgtcatag ccatggctga ggctacattc 60 aatgccatca acctcgaatt ccatgaaggt gctcaccctc gcttgggcgc actcgacgac 120 attatcttcc atccacttgg tcatgcgtcg ctcgacgagg cagcttggct tgccaaagca 180 gtggcagcag acattggcaa ccgattcagt gtgccagtgt ttctgtacgc cgcagcccac 240 ccaacaggga aggaaagttg atgccataag gcgagagctc ggatattacc ggccaaattc 300 aaggggaagt caatgggccg ggtgggcaat gcccgaaacg ctaccgctga gcctgatgaa 360 gggccaaacg tgggtttcaa gagctaaagg catcacantt gattgggtgc acgccccttg 420 ggnttacatt ctacaacgtt ccaatncctt tgcactgatg tgtcaant 468 12 128 PRT Glycine max UNSURE (87) Xaa can be any naturally ocurring amino acid 12 Gly Asn Pro Ile Tyr Ser Pro Leu His Gln Thr Val Ile Ala Met Ala 1 5 10 15 Glu Ala Thr Phe Asn Ala Ile Asn Leu Glu Phe His Glu Gly Ala His 20 25 30 Pro Arg Leu Gly Ala Leu Asp Asp Ile Ile Phe His Pro Leu Gly His 35 40 45 Ala Ser Leu Asp Glu Ala Ala Trp Leu Ala Lys Ala Val Ala Ala Asp 50 55 60 Ile Gly Asn Arg Phe Ser Val Pro Val Phe Leu Tyr Ala Ala Ala His 65 70 75 80 Pro Thr Gly Lys Glu Ser Xaa Cys His Lys Ala Arg Ala Arg Ile Leu 85 90 95 Pro Ala Lys Phe Lys Gly Lys Ser Met Gly Arg Val Gly Asn Ala Arg 100 105 110 Asn Ala Thr Ala Glu Pro Asp Glu Gly Pro Asn Val Gly Phe Lys Ser 115 120 125 13 416 DNA Triticum aestivum unsure (393) n is a, c, g or t 13 tcgaaggtga tctgttgcaa gctctacatc tctgaaagcc aaaatgcggc tgttgtcgat 60 gccatcagcc gcataggcca gaaagaccct gaggtggttc tactcaacaa gttcgaggat 120 gagtactaca accgtgtccg ctacacgctt gtctcctaca tcaccaacga aagctcgact 180 ggtggagctg tatttagccc aatcaggaag gtactgctgg cgatgatcga ggctgcattt 240 tcagccataa acctcgaagt gcactgtgga actcatccaa ggattggtgt cgtcgatgac 300 atttcattcc accccttgaa tcaagcggac acaatagagg atgctgctca gctggtaagc 360 tggtacctct gacattggaa tggttcaatt cantggtncc aaaangcgga acaata 416 14 302 PRT Triticum aestivum 14 Ser Lys Val Ile Cys Cys Lys Leu Tyr Ile Ser Glu Ser Gln Asn Ala 1 5 10 15 Ala Val Val Asp Ala Ile Ser Arg Ile Gly Gln Lys Asp Pro Glu Val 20 25 30 Val Leu Leu Asn Lys Phe Glu Asp Glu Tyr Tyr Asn Arg Val Arg Tyr 35 40 45 Thr Leu Val Ser Tyr Ile Thr Asn Glu Ser Ser Thr Gly Gly Ala Val 50 55 60 Phe Ser Pro Ile Arg Lys Val Leu Leu Ala Met Ile Glu Ala Ala Phe 65 70 75 80 Ser Ala Ile Asn Leu Glu Val His Cys Gly Thr His Pro Arg Ile Gly 85 90 95 Val Val Asp Asp Ile Ser Phe His Pro Leu Asn Gln Ala Asp Thr Ile 100 105 110 Glu Asp Ala Ala Gln Leu Ala Lys Leu Val Ala Ser Asp Ile Gly Asn 115 120 125 Gly Leu Gln Val Pro Val Phe Leu Tyr Ala Ala Ala His Pro Thr Ser 130 135 140 Lys Ser Val Ser Ala Val Arg Arg Glu Leu Gly Tyr Phe Arg Pro Asn 145 150 155 160 His Lys Gly Val Gln Trp Ala Gly Pro Val Leu Pro Asp Thr Leu Pro 165 170 175 Met Lys Pro Asp Val Gly Pro Val His Val Pro Arg Glu Arg Gly Ala 180 185 190 Thr Met Val Gly Ala Gln Pro Leu Val Glu Ser Tyr Asn Val Pro Ile 195 200 205 Phe Cys Lys Asp Val Pro Thr Val Arg Arg Ile Thr Arg Arg Val Thr 210 215 220 Gly Arg Ser Gly Gly Leu Pro Ser Val Gln Ala Leu Ala Leu Phe His 225 230 235 240 Gly Asp Asn Cys Thr Glu Ile Ala Cys Phe Leu Leu Asp Pro Asp His 245 250 255 Val Gly Ala Asp Arg Val Gln Trp Leu Val Glu Gln Ile Ala Glu Glu 260 265 270 Gln Gly Leu Glu Val Glu Lys Gly Tyr Phe Thr Asp Leu Ser Lys His 275 280 285 Met Met Leu Glu Arg Tyr Ser Glu Met Val Ser Ala Ala Asp 290 295 300 15 1076 DNA Zea mays 15 gcacgagcca agaaccacag ccaccgcgat ggcgcagatc atcgatggca aggccatcgc 60 cgccgacgtc cgccgcgagg tcgccgccga tgtggccgcg ctctcgtcgg cccacggact 120 cgtgccgggg ctggccgtgg tcatcgtggg gagcaggaag gactcgcaga cgtacgtgaa 180 catgaagcgc aaggcgtgcg ccgaggtcgg catctgctcc atcgacgtcg acctcccgga 240 ggacatctcc gagaccgcgc tcgtcgccga ggttcatcgc ctcaacgctg accccgcagt 300 gcacgggatc cttgtccagc ttccacttcc taagcatatc aacgaagaga agatactgag 360 cgagatttcc atcgagaaag atgtggatgg cttccatcct ctcaacattg gcaagcttgc 420 aatgaaaggc agagagccac tgttcgtacc atgtacgcca aaggggtgca tggagctctt 480 gtcaaggagc ggagtcactg ttaaaggtaa gcgggcagtt gtggttggtc gcagcaacat 540 cgtcgggcta cctgtatccc tgctccttct gaaggcagat gcgaccgtat ctgttgtgca 600 ctcgcggacc cctgatcctg aaagcattgt acgcgaagct gacatagtca tcgcggcagc 660 tgggcaggct atgatgatca aaggtgactg gatcaagcca ggtgctgcgg tcatcgatgt 720 cgggacgaac tccatcgatg accctacccg gaagtccggg gtacggctcg tcggcgatgt 780 ggatttcgca gcggcgagca aggttgctgg gtacctgact ccggttcccg gaggcgttgg 840 cccaatgacg gtggcaatgc tgctgaagaa cacggtggat ggggcaaagc gggggatagt 900 cgagtagcta cgttcatctc acttcacgtt gctgtacggc ctgtgttgca aggatgtgag 960 ctgactcgaa aagcgtgtgt tggttggtga acaatctgtt tcccaagaat aagaatgata 1020 gtcacagctg ttttcctgtt taataaatgc aatgaagaaa gaattttggc tttaaa 1076 16 290 PRT Zea mays 16 Met Ala Gln Ile Ile Asp Gly Lys Ala Ile Ala Ala Asp Val Arg Arg 1 5 10 15 Glu Val Ala Ala Asp Val Ala Ala Leu Ser Ser Ala His Gly Leu Val 20 25 30 Pro Gly Leu Ala Val Val Ile Val Gly Ser Arg Lys Asp Ser Gln Thr 35 40 45 Tyr Val Asn Met Lys Arg Lys Ala Cys Ala Glu Val Gly Ile Cys Ser 50 55 60 Ile Asp Val Asp Leu Pro Glu Asp Ile Ser Glu Thr Ala Leu Val Ala 65 70 75 80 Glu Val His Arg Leu Asn Ala Asp Pro Ala Val His Gly Ile Leu Val 85 90 95 Gln Leu Pro Leu Pro Lys His Ile Asn Glu Glu Lys Ile Leu Ser Glu 100 105 110 Ile Ser Ile Glu Lys Asp Val Asp Gly Phe His Pro Leu Asn Ile Gly 115 120 125 Lys Leu Ala Met Lys Gly Arg Glu Pro Leu Phe Val Pro Cys Thr Pro 130 135 140 Lys Gly Cys Met Glu Leu Leu Ser Arg Ser Gly Val Thr Val Lys Gly 145 150 155 160 Lys Arg Ala Val Val Val Gly Arg Ser Asn Ile Val Gly Leu Pro Val 165 170 175 Ser Leu Leu Leu Leu Lys Ala Asp Ala Thr Val Ser Val Val His Ser 180 185 190 Arg Thr Pro Asp Pro Glu Ser Ile Val Arg Glu Ala Asp Ile Val Ile 195 200 205 Ala Ala Ala Gly Gln Ala Met Met Ile Lys Gly Asp Trp Ile Lys Pro 210 215 220 Gly Ala Ala Val Ile Asp Val Gly Thr Asn Ser Ile Asp Asp Pro Thr 225 230 235 240 Arg Lys Ser Gly Val Arg Leu Val Gly Asp Val Asp Phe Ala Ala Ala 245 250 255 Ser Lys Val Ala Gly Tyr Leu Thr Pro Val Pro Gly Gly Val Gly Pro 260 265 270 Met Thr Val Ala Met Leu Leu Lys Asn Thr Val Asp Gly Ala Lys Arg 275 280 285 Gly Ile 290 17 579 DNA Oryza sativa unsure (316) n is a, c, g or t 17 gtttaaacgc ggcggcggcg gcgcctcctc taccgactca gatgcttgtg ccaagatcat 60 tgatgggaag ttggtggcaa agcagataag agaggaaatc gctgttgaga tcgccaagat 120 gaaggatgca attggggttg tgcctgggct ggcagtcatc ctagttgggt caaggaagga 180 ttctcaaacg tatgtgcgca acaagaagaa ggcatgcgaa gcggttggta tcaagtcata 240 tgaggttaat ttgccggaag acagctctga ggatgaggtt ctcaagcaca tcgcaacatt 300 taacagtgat ccgtcngtgc atggcatctt ggtcagttcc cctacctcat catatgaatg 360 atgagaacat tttgaatgct gtagtattga gaaggatgtt gatggattca ncactgaaca 420 ttggcgactg catgcaagcc ggatcgtcct tgtccagcac cctaagatca tggatacacc 480 agatatggan tgaatcaagg aanaactttg tattggcggg nattttggga tctgcgctat 540 acgcaaanca ccacgtacat gnatcaatca gaaccggga 579 18 292 PRT Oryza sativa 18 Ala Lys Ile Ile Asp Gly Lys Leu Val Ala Lys Gln Ile Arg Glu Glu 1 5 10 15 Ile Ala Val Glu Ile Ala Lys Met Lys Asp Ala Ile Gly Val Val Pro 20 25 30 Gly Leu Ala Val Ile Leu Val Gly Ser Arg Lys Asp Ser Gln Thr Tyr 35 40 45 Val Arg Asn Lys Lys Lys Ala Cys Glu Ala Val Gly Ile Lys Ser Tyr 50 55 60 Glu Val Asn Leu Pro Glu Asp Ser Ser Glu Asp Glu Val Leu Lys His 65 70 75 80 Ile Ala Thr Phe Asn Ser Asp Pro Ser Val His Gly Ile Leu Val Gln 85 90 95 Leu Pro Leu Pro His His Met Asn Asp Glu Asn Ile Leu Asn Ala Val 100 105 110 Ser Ile Glu Lys Asp Val Asp Gly Phe His Pro Leu Asn Ile Gly Arg 115 120 125 Leu Ala Met Gln Gly Arg Asp Pro Phe Phe Val Pro Cys Thr Pro Lys 130 135 140 Gly Cys Met Glu Leu Leu His Arg Tyr Gly Val Glu Ile Lys Gly Lys 145 150 155 160 Arg Ala Val Val Ile Gly Arg Ser Asn Ile Val Gly Met Pro Ala Ala 165 170 175 Leu Leu Leu Gln Lys Ala Asn Ala Thr Val Ser Ile Val His Ser Asn 180 185 190 Thr Lys Lys Pro Glu Glu Ile Thr Arg Gln Ala Asp Ile Val Ile Ala 195 200 205 Ala Val Gly Val Ala Asn Leu Val Arg Gly Ser Trp Ile Lys Pro Gly 210 215 220 Ala Ala Ile Ile Asp Val Gly Ile Asn Pro Val Asp Asp Pro Glu Ser 225 230 235 240 Pro Arg Gly Tyr Arg Leu Val Gly Asp Val Cys Tyr Glu Glu Ala Ser 245 250 255 Lys Ile Ala Gly Leu Ile Thr Pro Val Pro Gly Gly Val Gly Pro Met 260 265 270 Thr Ile Ala Met Leu Leu Ser Asn Thr Leu Glu Ser Ala Lys Arg Ile 275 280 285 His Lys Phe Lys 290 19 926 DNA Glycine max 19 gcagagagaa gaaggcttgt gaatctgttg gaatcaattc tttggaagca aatctgcctg 60 agaattccac agaagaagaa gttttgaact atattgcagg ctacaatgat gatccttcag 120 ttcatggcat cctcgttcag ttacccttac cttcgcatat gaatgagcag aacatcttga 180 acgctgtcag gattgagaag gatgtagatg gttttcatcc gttaaatatt ggtcgtcttg 240 ccatgcgtgg aagagaacct ctgtttgttc cttgtacacc aaagggatgc atagagctac 300 tgcacagata caatgtttct attaaaggaa agagggctgt tgtgattggt cggagcaata 360 ttgttggaat gccagctgct ctcttgcttc aaagggaaga tgctactgtc agtattgtcc 420 attctagaac cagtaacccc gaagagatca taagacaggc agatattatc attgctgctg 480 ttgggcaagc aaacatggtg aggggaagct ggataaagcc tggtgcagtc attattgatg 540 ttggaatcaa cccggtagag gatccaaata gcccccgagg ttacaaactg gtgggagatg 600 tttgttatga agaagccata agaattgcct ctgctgttac accagttcct ggaggagttg 660 gtccaatgac catagcaatg cttctacaaa atacactcat ctctgcaaag agggtgcaca 720 attttgaata acattgtgaa agggtgttgt ataccattat gagccatcaa tttttgttta 780 ggtgactcgt ggatttaagg tagggttttt tcaacattgg gacttaagcc ccaaataaga 840 gaaaatgttg ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaca aaaaaaaaaa 900 acttgagggg gccccggacc caatat 926 20 242 PRT Glycine max 20 Arg Glu Lys Lys Ala Cys Glu Ser Val Gly Ile Asn Ser Leu Glu Ala 1 5 10 15 Asn Leu Pro Glu Asn Ser Thr Glu Glu Glu Val Leu Asn Tyr Ile Ala 20 25 30 Gly Tyr Asn Asp Asp Pro Ser Val His Gly Ile Leu Val Gln Leu Pro 35 40 45 Leu Pro Ser His Met Asn Glu Gln Asn Ile Leu Asn Ala Val Arg Ile 50 55 60 Glu Lys Asp Val Asp Gly Phe His Pro Leu Asn Ile Gly Arg Leu Ala 65 70 75 80 Met Arg Gly Arg Glu Pro Leu Phe Val Pro Cys Thr Pro Lys Gly Cys 85 90 95 Ile Glu Leu Leu His Arg Tyr Asn Val Ser Ile Lys Gly Lys Arg Ala 100 105 110 Val Val Ile Gly Arg Ser Asn Ile Val Gly Met Pro Ala Ala Leu Leu 115 120 125 Leu Gln Arg Glu Asp Ala Thr Val Ser Ile Val His Ser Arg Thr Ser 130 135 140 Asn Pro Glu Glu Ile Ile Arg Gln Ala Asp Ile Ile Ile Ala Ala Val 145 150 155 160 Gly Gln Ala Asn Met Val Arg Gly Ser Trp Ile Lys Pro Gly Ala Val 165 170 175 Ile Ile Asp Val Gly Ile Asn Pro Val Glu Asp Pro Asn Ser Pro Arg 180 185 190 Gly Tyr Lys Leu Val Gly Asp Val Cys Tyr Glu Glu Ala Ile Arg Ile 195 200 205 Ala Ser Ala Val Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Ile 210 215 220 Ala Met Leu Leu Gln Asn Thr Leu Ile Ser Ala Lys Arg Val His Asn 225 230 235 240 Phe Glu 21 875 DNA Triticum aestivum unsure (584) n is a, c, g or t 21 ctctagtgct accccaatgg cgcaaatcat cgacggcaag gccatcgccg ccgaaatcag 60 gcgcgagatc ggcgccgagg tcgccgtgct ctcgtccgcc cacaacatcg tgccggggct 120 ggcggtggtg atcgtgggga gcaggaagga ctcgcagacg tacgtgcaga tgaagcgcaa 180 ggcctgcgcc gaggtcggca tccgctcctt cgacgtcgac ctccccgagg acatcgccga 240 ggccgcgctc gtcgccgagg tccaccgcct caacgccgac cccgccgtcc acggaattct 300 tgttcagctt ccattgccca agcatatcaa cgaagaaaat atcttaaacc agatctccat 360 tgagaaagat gtcgacggct ttcatccttt gaacattggc aagcttgcaa tgaaaggcag 420 agatccactg ttcgtacctt gcacgccaaa gggatgcatg gagctcctgt cacgaagtgg 480 cgtcactgta aaaggaaaac acgcagttgt ggttgggcgt agcaacatcg tgggtttacc 540 aagtatccct tctccttctg aaagcggacg ctaccgtgtc gatngtgcat caacggaccc 600 aaatccccaa acaatttccg tcaagcaaga cattgtcatt gcagcagctg ggcaagccat 660 gatgatcaag ggagactggn ttaaacaaaa gcgcaacgnc atcnacgtcg ggacaatcca 720 tcgacgacca acaagaatct gggtaaaatc cttggnagtg gttctcngag naacaagccn 780 ggtcactgat cggcccggan gntcggccat actggnattt ctaaaaaagg gganggncaa 840 angganncac gattcgcnna ttgaagggan attna 875 22 289 PRT Triticum aestivum UNSURE (195) Xaa can be any naturally ocurring amino acid 22 Ser Ser Ala Thr Pro Met Ala Gln Ile Ile Asp Gly Lys Ala Ile Ala 1 5 10 15 Ala Glu Ile Arg Arg Glu Ile Gly Ala Glu Val Ala Val Leu Ser Ser 20 25 30 Ala His Asn Ile Val Pro Gly Leu Ala Val Val Ile Val Gly Ser Arg 35 40 45 Lys Asp Ser Gln Thr Tyr Val Gln Met Lys Arg Lys Ala Cys Ala Glu 50 55 60 Val Gly Ile Arg Ser Phe Asp Val Asp Leu Pro Glu Asp Ile Ala Glu 65 70 75 80 Ala Ala Leu Val Ala Glu Val His Arg Leu Asn Ala Asp Pro Ala Val 85 90 95 His Gly Ile Leu Val Gln Leu Pro Leu Pro Lys His Ile Asn Glu Glu 100 105 110 Asn Ile Leu Asn Gln Ile Ser Ile Glu Lys Asp Val Asp Gly Phe His 115 120 125 Pro Leu Asn Ile Gly Lys Leu Ala Met Lys Gly Arg Asp Pro Leu Phe 130 135 140 Val Pro Cys Thr Pro Lys Gly Cys Met Glu Leu Leu Ser Arg Ser Gly 145 150 155 160 Val Thr Val Lys Gly Lys His Ala Val Val Val Gly Arg Ser Asn Ile 165 170 175 Val Gly Leu Pro Ser Ile Pro Ser Pro Ser Glu Ser Gly Arg Tyr Arg 180 185 190 Val Asp Xaa Ala Ser Thr Asp Pro Asn Pro Gln Thr Ile Ser Val Lys 195 200 205 Gln Asp Ile Val Ile Ala Ala Ala Gly Gln Ala Met Met Ile Lys Gly 210 215 220 Asp Trp Xaa Lys Gln Lys Arg Asn Xaa Ile Xaa Val Gly Thr Ile His 225 230 235 240 Arg Arg Pro Thr Arg Ile Trp Val Lys Ser Leu Xaa Val Val Leu Xaa 245 250 255 Xaa Thr Ser Xaa Val Thr Asp Arg Pro Gly Xaa Ser Ala Ile Leu Xaa 260 265 270 Phe Leu Lys Lys Gly Xaa Xaa Lys Xaa Xaa His Asp Ser Xaa Ile Glu 275 280 285 Gly 

What is claimed is:
 1. An isolated nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:2; (b) an isolated nucleic acid fragment that is complementary to (a).
 2. The isolated nucleic acid fragment of claim 1 wherein nucleic acid fragment is a functional RNA.
 3. The isolated nucleic acid fragment of claim 1 wherein the nucleotide sequence of the fragment comprises the sequence set forth in SEQ ID NO:
 1. 4. A chimeric gene comprising the nucleic acid fragment of claim 1 operably linked to suitable regulatory sequences.
 5. A transformed host cell comprising the chimeric gene of claim
 4. 6. A 3-methyl-2-oxobutanoate hydroxymethyltransferase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2.
 7. An isolated nucleic acid fragment encoding a formyltetrahydrofolate deformylase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4, 6 and 8; (b) an isolated nucleic acid fragment that is complementary to (a).
 8. The isolated nucleic acid fragment of claim 7 wherein nucleic acid fragment is a functional RNA.
 9. The isolated nucleic acid fragment of claim 7 wherein the nucleotide sequence of the fragment comprises the sequence set forth in SEQ ID NO:3, 5 and
 7. 10. A chimeric gene comprising the nucleic acid fragment of claim 7 operably linked to suitable regulatory sequences.
 11. A transformed host cell comprising the chimeric gene of claim
 10. 12. A formyltetrahydrofolate deformylase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, 6 and
 8. 13. An isolated nucleic acid fragment encoding a glutamate formiminotransferase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:10, 12 and 14; (b) an isolated nucleic acid fragment that is complementary to (a).
 14. The isolated nucleic acid fragment of claim 13 wherein nucleic acid fragment is a functional RNA.
 15. The isolated nucleic acid fragment of claim 13 wherein the nucleotide sequence of the fragment comprises the sequence set forth in SEQ ID NO:9, 11 and
 13. 16. A chimeric gene comprising the nucleic acid fragment of claim 13 operably linked to suitable regulatory sequences.
 17. A transformed host cell comprising the chimeric gene of claim
 16. 18. A glutamate formiminotransferase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10, 12 and
 14. 19. An isolated nucleic acid fragment encoding a methylenetetrahydrofolate dehydrogenase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:16, 18, 20 and 22; (b) an isolated nucleic acid fragment that is complementary to (a).
 20. The isolated nucleic acid fragment of claim 19 wherein nucleic acid fragment is a functional RNA.
 21. The isolated nucleic acid fragment of claim 19 wherein the nucleotide sequence of the fragment comprises the sequence set forth in SEQ ID NO:15, 17, 19 and
 21. 22. A chimeric gene comprising the nucleic acid fragment of claim 19 operably linked to suitable regulatory sequences.
 23. A transformed host cell comprising the chimeric gene of claim
 22. 24. A methylenetetrahydrofolate dehydrogenase polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16, 18, 20 and
 22. 